# US 2014/0154696 A1 · Pcr diagnostics of dermatophytes and other pathogenic fungi > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0154696 A1 - Document ID: US20140154696A1 - Status: Published application - Kind: A1 - Application number: 14/175,853 - Filed: 2014-02-07 - Published: 2014-06-05 - Earliest claimed: 2005-06-14 - Assignees: Statens Serum Institut - Applicants: Statens Serum Institut - Inventors: Anna H. Brillowska-Dabrowska - Web page: https://patentgrep.com/patent/US20140154696A1 ## CPC - C12Q 1/6895: for plants, fungi or algae - C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor} - C12Q 2600/16: Primer sets for multiplex assays - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Dermatophytes which belong to one of the three genera Epidermophyton, Trichophyton and Microsporum are the main cause of fungal infections of skin, hair and nails. Traditional diagnostic procedures consist of microscopy and culture, but due to the slow growth rate of dermatophytes typically two to four weeks are needed before a final diagnosis is obtained. The present invention is a rapid DNA extraction method extracting nucleic acids from fungi (e.g. dermatophytes and other pathogenic fungi) which can be performed from directly on hair, nail or skin specimens from humans, from naturally or experimentally infected animals or from cultured fungal colonies for the use in PCR amplification and detection assays. The present invention also includes specific primer sets for detection of any dermatophyte and for species specific detection of Trichophyton rubrum and Epidermophyton floccosum by PCR and a kit for diagnosing fungal infections. ## Claims ### Claim 1 A method of extracting nucleic acid from fungi comprising the following steps: (a) heating a sample containing the fungi in a lysis buffer to between 80 and 100° C. for 10 minutes; and (b) mixing the solution with a neutralizing buffer; wherein said lysis buffer consists of, in aqueous solution: (i) a reducer selected from the group consisting of sodium carbonate, sodium sulfate, beta-mercaptoethanol, dithiothreitol, sodium sulfide, sodium chloride, sodium chlorite, sodium iodate, and sodium bicarbonate; (ii) a salt selected from the group consisting of potassium chloride and ammonium persulfate; and (iii) a buffering compound selected from the group consisting of CAPSO, TRIS, HEPES, and phosphate buffer; wherein said neutralizing buffer is an aqueous 0.5-3% w/v protein solution; and wherein the cell wall of said fungi is not disrupted prior to step (a). ### Claim 2 (depends on 1) The method according to claim 1, wherein said fungi is a species belonging to the genera Trycophyton, Epidermophyton, or Microsporum, or a yeast. ### Claim 3 (depends on 1) The method according to claim 1, wherein said sample is a patient sample. ### Claim 4 (depends on 3) The method according to claim 3, wherein said patient sample is selected from the group consisting of hair, skin, and nail. ### Claim 5 (depends on 1) The method according to claim 1, wherein said sample is cultured fungi. ### Claim 6 (depends on 1) The method according to claim 1, wherein said lysis buffer consists of sodium bicarbonate, potassium chloride, and TRIS. ### Claim 7 (depends on 1) The method according to claim 1, wherein said sample in step (a) is heated to 95° C. for 10 minutes. ### Claim 8 (depends on 1) The method according to claim 1, wherein the neutralizing buffer is an aqueous 2% w/v protein solution ### Claim 9 (depends on 1) The method according to claim 1, wherein the protein in said neutralizing buffer is BSA or ovalbumin. ### Claim 10 A method of detecting fungi infections in patient samples comprising the following steps: (A) extracting nucleic acid from the sample by: (a) heating a sample containing the fungi in a lysis buffer to between 80 and 100° C. for 10 minutes; and (b) mixing the solution with a neutralizing buffer; wherein said lysis buffer consists of, in aqueous solution: (i) a reducer selected from the group consisting of sodium carbonate, sodium sulfate, beta-mercaptoethanol, dithiothreitol, sodium sulfide, sodium chloride, sodium chlorite, sodium iodate, and sodium bicarbonate; (ii) a salt selected from the group consisting of potassium chloride and ammonium persulfate; and (iii) a buffering compound selected from the group consisting of CAPSO, TRIS, HEPES, and phosphate buffer; wherein said neutralizing buffer is an aqueous 0.5-3% w/v protein solution; (B) amplifying the nucleic acid with PCR using fungus-specific primers; and (C) detecting fungus-specific genes. ### Claim 11 (depends on 10) The method according to claim 10, wherein said fungus-specific primers are selected from the group consisting of panDerm1 (SEQ ID NO: 1), panDerm2 (SEQ ID NO: 2), uni (SEQ ID NO: 3), Trubrum-rev (SEQ ID NO: 4), Ef-rev (SEQ ID NO: 5), Micr773-for (SEQ ID NO: 10), Micr885-rev (SEQ ID NO: 11), Trichopyros-for (SEQ ID NO: 12), and Trichopyros-rev (SEQ ID NO: 13). ### Claim 12 A multiplex PCR kit for extracting nucleic acid from fungi and detecting dermatophytes comprising: (a) a lysis buffer consisting of, in aqueous solution: (i) a reducer selected from the group consisting of sodium carbonate, sodium sulfate, beta-mercaptoethanol, dithiothreitol, sodium sulfide, sodium chloride, sodium chlorite, sodium iodate, and sodium bicarbonate; (ii) a salt selected from the group consisting of potassium chloride and ammonium persulfate; and (iii) a buffering compound selected from the group consisting of CAPSO, TRIS, HEPES, and phosphate buffer; (b) a neutralizing buffer, wherein said neutralizing buffer is an aqueous 0.5-3% w/v protein solution; (c) fungus-specific primers selected from the group consisting of panDerm1 (SEQ ID NO: 1), panDerm2 (SEQ ID NO: 2), uni (SEQ ID NO: 3), Trubrum-rev (SEQ ID NO: 4), Ef-rev (SEQ ID NO: 5), Micr773-for (SEQ ID NO: 10), Micr885-rev (SEQ ID NO: 11), Trichopyros-for (SEQ ID NO: 12), and Trichopyros-rev (SEQ ID NO: 13); and (d) agents for performing a PCR reaction.